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Induction of gene expression using a high concentration sugar mixture

Inactive Publication Date: 2018-05-31
DANISCO US INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent is about improving the production of proteins from cell cultures by using a specific method to increase the amount of protein produced. This method involves inducing a cell culture with a specific mixture of sugars and enzymes to create an inducing feed composition. This inducing feed composition is then used to contact the cell culture to induce the expression of the protein of interest. The method can be used with various cell cultures and can produce various proteins, such as enzymes, hormones, growth factors, cytokines, and antibodies. Overall, this patent provides a way to increase protein production in cell cultures and improve the efficiency of protein production.

Problems solved by technology

In such parts where sucrose sources are abundant and inexpensive, the cost of glucose can be deemed relatively very expensive especially in situation where large amounts or volumes of such sugar source is required to produce an inducer for use in large, commercial scale operations.

Method used

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  • Induction of gene expression using a high concentration sugar mixture

Examples

Experimental program
Comparison scheme
Effect test

example 1

Glucose / Sophorose (G / S) Control Fermentation

[0178]The glucose / sophorose solution for induction of a control T. reesei protein production fermentation was prepared as previously described, in, for example, U.S. Pat. No. 7,713,725. The glucose / sophorose preparation was diluted to match the sugar solids content of the sucrose feeds. Solids content was 55%.

[0179]A typical industrial scale Trichoderma reesei fermentation process comprises two phases. First phase is the biomass growth phase, with little to no protein production; followed by the protein production phase, with little to no biomass growth. During the second phase, the rate of protein production is, in normal circumstances, linear. The protein production rate is the average rate from the start of the production phase to the end of the run. The yield calculation is the amount of protein produced during the protein production phase divided by the amount of sugars consumed during the same time period. Total protein in fermentati...

example 2

Sugarcane Juice Syrup (SJS) Fermentation

[0181]Sugarcane juice syrup (sucrose) was inverted and reverted, and the resulting syrup was fed to T. reesei protein production fermentations. The results were compared to the control fermentation of the same strain, fed glucose / sophorose.

[0182]Three replicate fermentations were run using inverted and reverted Sugarcane Juice Syrup. Inversion was carried out at 75° C. and pH 2.0 for 4 hrs, followed by reversion for 48 hours at pH 4.0 and 60° C. with T. reesei beta-glucosidase (Bgl1) included at a concentration of 64,000 U / kg syrup. Alternatively inversion may be carried out employing a suitable invertase enzymes, for example, one derived from Aspergillus niger (UniPro Accession Number: QOZR36), Aspergillus fumigates, Aspergillus japonicas, Aspergillus nidulans, or other Aspergillus spp, or one derived from Fusarium oxyporum, or other like fungal species, as well as from various bacterial or even plant sources. The inversion reaction may be ca...

example 3

Very High Purity Sucrose (VHP) Fermentation

[0185]Very high purity (VHP) sucrose was inverted and reverted, and the resulting sugars fed to T. reesei protein production fermentations. The results were compared to the control fermentation of the same strain, fed glucose / sophorose.

[0186]Duplicate fermentations of T. reesei using the inverted and reverted VHP sucrose feed were performed. Inversion was carried out at 75° C. and pH 2.0 for 4 hrs, followed by reversion for 48 hours at pH 4.0 and 60° C. with T. reesei beta-glucosidase (Bgl1) included at a concentration of 64,000 U / kg syrup.

[0187]The total solids content of the VHP feed was 55%. A control fermentation was run in which the VHP inversion was conducted, but the reversion was omitted.

[0188]One of the VHP duplicate fermentations showed contamination in the final two time point samples. This did not appear to have a major effect on the performance. Yield was reduced by 3% relative to the non-contaminated run and rate was reduced b...

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Abstract

Described herein is a composition useful for inducing expression of genes whose expression is under control of an inducible promoter sequence and methods for the compositions preparation and use.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The instant application claims priority to U.S. Provisional Patent Application Ser. No. 62 / 189,462, filed Jul. 7, 2015, which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]This invention relates to methods for improved production of proteins from a cell culture. The invention also pertains to culture components and conditions that increase the amount of protein produced from genes under the control of inducible gene promoter. The improved methods can be used for the production of proteins encoded by naturally occurring cellulase genes as well as from various heterologous constructs.BACKGROUND OF THE INVENTION[0003]Filamentous fungi and cellulolytic bacteria produce extracellular cellulase enzymes that confer on the organisms the ability to hydrolyze the β-(1,4)-linked glycosidic bonds of cellulose to produce glucose. These enzymes provide the organisms with the ability to use cellulose, the most abundant ...

Claims

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Application Information

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IPC IPC(8): C12N1/38C12P21/00
CPCC12N1/38C12P21/00C12P19/02C12P21/02
Inventor DODGE, TIMOTHY C.KRUUS, ILKKA ILARIMITCHINSON, COLINVILJAVA, TIMO TAPIO
Owner DANISCO US INC
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